The one-carbon metabolism as an underlying pathway for placental DNA methylation – a systematic review

ABSTRACT Epigenetic modifications, including DNA methylation, are proposed mechanisms explaining the impact of parental exposures to foetal development and lifelong health. Micronutrients including folate, choline, and vitamin B12 provide methyl groups for the one-carbon metabolism and subsequent DNA methylation processes. Placental DNA methylation changes in response to one-carbon moieties hold potential targets to improve obstetrical care. We conducted a systematic review on the associations between one-carbon metabolism and human placental DNA methylation. We included 22 studies. Findings from clinical studies with minimal ErasmusAGE quality score 5/10 (n = 15) and in vitro studies (n = 3) are summarized for different one-carbon moieties. Next, results are discussed per study approach: (1) global DNA methylation (n = 9), (2) genome-wide analyses (n = 4), and (3) gene specific (n = 14). Generally, one-carbon moieties were not associated with global methylation, although conflicting outcomes were reported specifically for choline. Using genome-wide approaches, few differentially methylated sites associated with S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), or dietary patterns. Most studies taking a gene-specific approach indicated site-specific relationships depending on studied moiety and genomic region, specifically in genes involved in growth and development including LEP, NR3C1, CRH, and PlGF; however, overlap between studies was low. Therefore, we recommend to further investigate the impact of an optimized one-carbon metabolism on DNA methylation and lifelong health.


Introduction
Foetal exposures within the intra-uterine environment can affect health outcomes of the offspring during the life course.This is known as the developmental origins of health and disease (DOHaD) paradigm [1,2].Epigenetic modifications can affect gene-expression and consequently cell function without changing the DNA sequence and are proposed underlying mechanisms connecting parental exposures to gamete maturation, embryonic and foetal development, and long-term health outcomes in the offspring [1,3].
The best characterized epigenetic mechanism is DNA methylation.DNA methylation is regulated by DNA methyltransferases (DNMTs), which transfer methyl groups predominantly to the C-5 position of a cytosine at a cytosine-phosphate-guanine (CpG) site [4,5].Methylation of CpGs in regulatory genomic regions like promoters typically leads to gene silencing while non-genic, repetitive DNA sequences, such as transposable elements (e.g., LINE-1 and Alu elements), are often heavily methylated to maintain genomic stability and can serve as markers for global methylation [4][5][6].
During the periconception period, starting 14 weeks before conception till 10 weeks after conception, significant epigenetic modifications with potential effects along the life course take place [7].Most DNA methylation marks present in parental gametes are removed during the first cell divisions in the zygote and blastocyst stages, followed by the establishment of de novo DNA methylation most prominent after implantation.In utero, proper de novo DNA methylation is essential for key biological processes involved in regulation of gene expression during development and differentiation [4][5][6][7][8].Therefore, the periconception and pregnancy period is a vulnerable time span for epigenetic disruptions.
The one-carbon metabolism provides methyl groups required amongst others for DNA methylation.Herein, methionine is converted to the major methyl donor s-adenosylmethionine (SAM), which in turn is converted to s-adenosylhomocysteine (SAH) thereby donating its methyl group.Other essential substrates of the one-carbon metabolism, here referred to as one-carbon moieties, include methyl donors such as folate and choline, and cofactors including vitamin B 2 , B 6 , and B 12 , that are all mostly derived from diet (Figure 1) [9].
Evidence from predominantly animal studies indicates that differences in one-carbon metabolism during pregnancy can cause lasting DNA methylation changes in the offspring [7,[10][11][12][13][14][15][16][17].A famous example is that a methyl-supplemented diet fed to pregnant dams of the Agouti mouse increases DNA methylation of a regulating locus that results in a lower expression of the Agouti gene in their offspring, resulting in changed adiposity and coat colour [18,19].In humans conceived during the Dutch Hunger Winter, an increased risk for obesity was observed and methylation differences in whole blood are found related to genes involved in growth and metabolism including lower methylation of insulin-like growth factor 2 (IGF2) gene [20].Additionally, offspring of women who used periconceptional folic acid supplements had a higher methylation of IGF2 in blood [15].This illustrates that nutrition, particularly moieties of one-carbon metabolism, plays a crucial role in early life epigenetic programming with potential lifelong health consequences [7].
The placenta is an interesting target organ for DNA methylation studies investigating the relationship between maternal exposures including nutrition, one-carbon metabolism, and foetal programming as it forms the unique and indispensable interface between the mother and the developing embryo and foetus [21].The changes observed in the placental DNA methylation are suggested to reflect alterations in placental functioning and as such impact foetal programming.The treatment of these adverse variations could provide targets for future preventative or therapeutic interventions (Figure 2) [22].Additionally, we suggest that the presence of placental-originated cell free DNA Dietary methionine is converted to homocysteine via SAM and SAH.SAM is a major methyl donor for biological processes, including DNA methylation.The remethylation of homocysteine to methionine depends on other moieties including vitamin B12 as cofactor and 5 methyl tetrahydrofolate as substrate.MTHFR = methylenetetrahydrofolate reductase, BHMT = betaine-homocysteine S-methyltransferase, MS = methionine synthase, SAM = s-adenosylmethionine, SAH = s-adenosylhomocysteine (cfDNA) in maternal blood will become an opportunity to non-invasively assess placental DNA methylation profiles during early pregnancy, whereas the non-invasive collection of foetal tissues is not possible.This emphasizes the potential of the placenta as a proxy for foetal health conditions [23].
Previous reviews about the impact of the onecarbon metabolism in reproduction have mainly focussed on DNA methylation in tissues other than placenta including umbilical cord blood, focussed on animal studies and/or were limited to one specific one-carbon moiety [7,[10][11][12][13][14][24][25][26].Additionally, epigenome-wide association studies have specifically emerged over the last years [27].Therefore, this systematic review aims to provide an overview of the current knowledge obtained from human studies investigating the impact of the one-carbon metabolism on placental DNA methylation.Epigenetic derangements in response to impaired one-carbon metabolism could hold potential targets for future therapeutic or preventative interventions, including prenatal diagnosis and obstetrical care.

Protocol and registration
The protocol for this systematic review was registered to the PROSPERO registry (PROSPERO 2023 CRD42023393358).The PRISMA Guidelines for systematic reviews and meta-analysis protocols were followed [28].

Search strategy
Medline (on the Ovid platform), Embase, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, and Google Scholar were searched until 12 July 2023.An expert research librarian was involved in setting up the search strategy.The search strategy included but was not limited to terms related to one-carbon metabolism like folate, homocysteine, and nutrition combined with placental related terms and key words related to epigenetics including DNA methylation.The full search is provided in Table S1.

Study selection
Clinical studies were eligible if they studied onecarbon moieties during the preconception period and/or during pregnancy including birth and studied DNA methylation in human placental tissues.Since in practice, one-carbon moieties are consumed by foods and vitamin supplements, we also included articles investigating associations between dietary patterns reflecting the intake of one-carbon moieties or intake of multivitamin supplements.In vitro studies were eligible if they studied one-carbon moieties and DNA methylation in a human in vitro placental model.Review articles, conference abstracts, and articles not available in English language were excluded.MV and SS performed the title and abstract screening and the full-text review of remaining articles.

Quality assessment of included studies
We used the ErasmusAGE quality score system for systematic reviews to assess the quality of included clinical studies [29].Quality of included articles was assessed by MV and SS independently.All articles were scored based on five items and each item was scored zero, one or two points, leading to a score between 0-10 for each article.These items include study design (cross-sectional study = 0, longitudinal study = 1, intervention study = 2), study size (<50 patients = 0, 50-100 patients = 1, >100 patients = 2), method of measuring exposure and method of measuring outcome (both: no appropriate measure = 0, moderate quality of measure = 1, adequate measure = 2), and adjustments for confounders (no adjustment = 0, adjustment for key confounders = 1, adjustment for additional covariates or extra confounders = 2) (Table S2) [29].We discussed all articles with an ErasmusAGE quality score of at least 5.

Data extraction
Data extraction was done using a predefined form.Collected data included: study design, country of origin, year of publication, study population including sample size, method of measuring (exposure to) one-carbon moieties, timing of exposure (e.g., preand periconceptional, pregnancy), which part of placental tissue was studied (e.g., foetal or maternal side biopsy), focus of DNA methylation (global, genome-wide, or gene-specific approach), specified methylation technique.Lastly the main outcomes of the study related to DNA methylation were collected.

Study selection
The literature search resulted in 1490 unique records after duplicate removal.After title and abstract screening, 1435 articles were excluded and a total of 55 full-text articles were assessed for eligibility of which 22 were included for the current review (Figure 3).The reasons for final exclusion included no exposure to or measurements of one-carbon moieties (n = 15), no placental tissue studied (n = 7), no DNA methylation as outcome (n = 5) and other (n = 6) (Figure 3).

Study characteristics
The study characteristics and main outcomes of included studies are provided in Table 1.The included clinical studies were observational (n = 16) or clinical intervention studies (n = 3).Two solely in vitro studies were included [50,51] and one observational study combined clinical and in vitro work [43].All included in vitro studies used human placental cell lines.
One-carbon moieties that were studied in the clinical studies were folate/folic acid, (n=9), vitamin B 12 (n=9), homocysteine (n=4), choline (n=2), SAM and SAH (n=3), combination supplements (n=4), and dietary patterns reflecting differences in intake of one-carbon moieties (n=1).Two in vitro studies investigated the effect of folate and one studied the effect of choline on DNA methylation (Table 1).Results are discussed below for the different included moieties.
Most clinical studies used a gene-specific approach (n=14/20).Four used a genome-wide approach and global DNA methylation was investigated in nine studies.Some studies used multiple approaches in the same cohort.Table 2 summarizes the studies that investigated the associations for all three approaches in clinical studies with an ErasmusAGE quality score of at least 5 points.Included clinical studies scored between 3 and 8 points (median 5) (Table S2).

Metabolic determinants of one-carbon metabolism and placental DNA methylation outcomes
Folate/Folic acid Dietary intake/supplement use.One study (ErasmusAGE score 6) assessed dietary folate intake during pregnancy by Food Frequency Questionnaires (FFQ) and found no association with methylation of partially-or highly methylated domains after controlling for the False Discovery Rate (FDR) [38].
Liu et al. (ErasmusAGE score 7) showed lower methylation of 5 out of 9 investigated sites within FAM198A in women who started using folic acid supplements before pregnancy in male offspring (p-values between 0.026 -0.050) but not in female offspring, indicating a sex-specific impact on DNA methylation.No associations were found between use of folic acid supplements and methylation of 2 ANKRD20B sites [35].FAM198A and ANKRD20B sites were investigated, after these were formerly identified as top-ranked differentially methylated positions between 3 small for gestational age (SGA) children and 3 appropriate growth for gestational age (AGA) controls.Loke et al. (ErasmusAGE score 6) showed lower methylation of the differentially methylated region (DMR) of the H19 promoter in women using folic acid supplements in first trimester compared to no use of folic acid supplement, but found no methylation differences in the IGF2/H19 imprinted control region (ICR) or two IGF2 DMRs [36].

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Women delivering preterm had lower plasma folate and higher homocysteine compared to term controls.Mean MTR promoter methylation was 0.5% higher (p< 0.01) in preterm births.5/7 studied CpGs had higher methylation in preterm placentas only after vaginal deliveries.

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No associations between homocysteine or folate and DNA methylation were investigated.

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No association between vitamin B 12 or homocysteine and DNA methylation was studied.

3
In vitro studies using human placental cell lines Jiang (2016) -USA [50] In vitro study regions between 200 and 500 base pairs with >50% CG content, the 0-2 kb and 2-4kb up-and downstream regions next to CpG-islands, referred to as 'shores' and 'shelves' respectively, and CpGs outside these regions referred to as 'open sea.' Additionally, they took a genome-wide approach investigating single CpGs.The mother-infant dyads in their study were either homozygous for the wild-type MTHFR677 genotype or for the MTHFR C677T variant.The MTHFR C667T polymorphism leads to reduced methylenetetrahydrofolate reductase (MTHFR) activity which is involved in the one-carbon metabolism, leading to higher homocysteine and lower SAM concentrations (Figure 1).In the total study population, red blood cell (RBC) folate was positively associated with methylation of 'shelve' regions and CpG island.In mother-infant dyads homozygous for the MTHFR C677T variant, a positive association was also found between RBC folate and methylation of 'open sea' and 'shore' regions.No associations between individual CpGs and RBC folate remained significant after correcting for multiple testing [42].
In vitro studies.Investigation of DNA methylation in JEG-3, a placental choricocarcinomaderived cell line and in HTR-8/SVneo cell line, a model of extra-villous trophoblast, in response to folic acid treatment showed no changes in LINE-1 methylation [43].In contrast, global methylation decreased in both cell lines in a dosedependent manner with the largest effect in HTR-8/SVneo cells, where a 4.3% decrease (p < 0.001) was found at 10 −7 M folic acid, comparable to the physiological range of 400-600 µg/day, and a 5.7% decrease (p<0.001) was found at a much higher concentration of 10 −4 M folic acid.Taking a genespecific approach, promoter methylation of the tumour suppressor gene APC increased in both cell lines in response to folic acid, while for P16 an increase was observed in JEG-3 cells while methylation decreased in HTR-8/SVneo cells.Methylation of the promoter regions of oncogenes c-myc and c-jun decreased in both cell lines [43].
No changes were found in the MMP-9 promoter region.In a comparable in vitro study, methylation in the promoter regions of SNRPN, PEG10, and MEST was studied [51].Folic acid suppletion decreased methylation of SNRPN in a dosedependent manner in JEG-3, HTR-8/SVneo cell lines and in cytotrophoblasts.No changes were found for PEG10 and MEST [51].

Interim conclusions -Folate/folic acid
Mixed results have been reported for the relationship between folate/folic acid and markers of global placental DNA methylation.Only one study took a genome-wide approach and identified no significant associations between methylation of single CpGs and cord blood folate.When taking a gene-specific approach, several -but not all -investigated sites associated with folate/folic acid (Table 2).This indicates site-specific relationships between DNA methylation and folate/ folic acid.[37].In vitro studies.In an in vitro study using BeWo cells as a model for first trimester trophoblast, global DNA methylation was not altered by choline treatment [50].

Interim conclusions -Choline
Mixed results have been reported for choline and markers of global placental DNA methylation as well as for choline and NR3C1 methylation.Several other relationships between DNA methylation of specific genes and choline intake or blood levels were found depending on studied site and moment of measuring choline, indicating sitespecific relationships between DNA methylation and choline which may differ depending on timing of exposure (Table 2).[39].

Interim conclusions -SAM/SAH
A higher SAM:SAH ratio indicates higher availability of methyl group donors (Figure 1).Although only two studies investigated SAM and SAH, the SAM:SAH ratio in umbilical cord blood associated positively with markers of global methylation depending on genotype, and SAM, SAH, and SAM:SAH associated with methylation of a few individual CpGs in a genome-wide study.Both placental SAM and SAM:SAH ratio associated positively with PlGF methylation (Table 2).This suggests that SAM and SAH can impact placental DNA methylation both at a regional/global level as well as at gene-specific sites.Methylation of 2 CpGs in the IGF1 promoter region were assessed.No differences were found between study groups [33].Composition of prenatal vitamins varies but they usually contain folic acid, vitamin B 6 , and B 12 as well as other micronutrients.Lecorguille et al. (ErasmusAGE score 7) found higher methylation of Alu repetitive elements in women taking vitamin supplements before and during pregnancy compared to women not taking any vitamins (β = 0.40, p = 0.005) although no differences in LINE-1 methylation were found.Vitamin use only before or during pregnancy was not associated with Alu or LINE-1 methylation [32].

Combinations
Two studies investigated placental DNA methylation and self-reported maternal vitamin intake in the first month of pregnancy.Dou et al. (ErasmusAGE score 8) studied DNA methylation in the MARBLES and in the EARLI cohort, both comprise couples at increased risk for offspring with autism spectrum disorder (ASD).Mean array methylation for MARBLES women taking vitamins was −0.60% (CI −1.08, − 0.13) and the same magnitude of effect was seen in the EARLI cohort, although not significant −0.52% (CI −1.04, 0.01).No individual CpGs associated with vitamin intake using the FDR-corrected significance threshold (p < 1.0 −7 ).With a less stringent significance threshold of p < 0.01, they found 9216 and 3442 differentially methylated CpGs in the MARBLES and EARLI cohort, respectively.About 95% of these showed lower methylation in the vitamin group in both cohorts (average −4%).The identified CpGs were enriched in neuronal developmental pathways [31].Schmidt et al. (ErasmusAGE score 6) found no associations between vitamin use and methylation of predefined partially-or highly methylated domains or methylation of enhancers, active promoters, and bivalent promoters [38].

Interim conclusions -combinations
Global methylation was not associated with dietary patterns reflecting differences in intake of onecarbon moieties and use of vitamin supplements showed both increased and decreased global methylation as well as no differences depending on used technique and/or timing of supplement use.A genome-wide study revealed a few DMRs associated with dietary patterns, but dietary patterns or vitamin supplement use did not associate with individual CpGs after FDR correction.IGF1 promoter methylation was not altered in women taking SQLNS (Table 2).In general, dietary patterns and vitamin supplement use did not substantially impact placental DNA methylation in included studies.

Discussion
This review systematically summarizes associations between the one-carbon metabolism and placental DNA methylation.Included studies suggest that one-carbon moieties can impact placental DNA methylation at multiple specific genomic regions rather than affecting global methylation.On the other hand, this review shows that there is a lot of heterogeneity among studies regarding investigated exposures, studied outcomes, sample size, and study design.Additionally, differences in DNA methylation have been found between female and male offspring [35], depending on which part of the placenta was sampled [52], timing of exposure [32,37], and underlying genotype [42], which could also explain parts of observed differences between studies.Based on the use of ErasmusAGE quality score, we discussed studies with a quality score of at least 5 out of 10 points.

Global Methylation
A wide variety of techniques to study global methylation have been used by included studies (Table 1).Global DNA methylation was not altered by the use of vitamin B12 supplements, while higher intake of choline did increase global DNA methylation.One study found associations between one-carbon moieties and average DNA methylation to differ between genomic regions (i.e., CpG islands, 'shores,' 'shelves' and 'open sea') [42].Overall, most studies found no associations between one-carbon moieties and measures of global DNA methylation (Table 2).Hence, higher availability of methyl donors provided by the one-carbon metabolism does not just lead to increased global placental methylation.

Genome-wide approach
The number of included genome-wide studies is limited, and only three had a quality score of at least five.All three studies used the Illumina Infinium HumanMethylation450 (450K) BeadChip array, which covers >480,000 CpGs and one partly used the Infinium methylation EPIC array covering >850,000 CpGs.All three studies investigated different exposures (Table 2).CpGs associated with maternal vitamin use in the first month of pregnancy could only be identified when a less stringent FDR-corrected p-value was applied and 95% of these were hypomethylated in women who did take vitamins in the first month of pregnancy.Identified CpGs were enriched in neuronal developmental pathways [31].A few DMRs linked to different genes could be identified based on dietary patterns reflecting differences in intake of onecarbon moieties [32] and methylation of a few CpGs associated with umbilical cord levels of SAM, SAH, and SAM:SAH, but not with RBC folate or vitamin B 12 levels in cord blood [42].

Gene-specific approach
Included studies mostly investigated different loci, hampering comparison of results.Overall, most gene-specific studies did find relationships between one-carbon moieties and placental DNA methylation.On the other hand, most studies reported both significant and non-significant associations and there is a lack of uniformity in directionality of identified associations, indicating sitespecific relationships (Table 2).

Genes involved in growth and metabolism
Genes involved in growth and metabolism have been of particular interest in included studies.
The imprinted IGF2/H19 cluster plays an important role in foetal and placental growth [53].Methylation of IGF2/H19 has been investigated in multiple studies, with most reporting no significant associations with investigated onecarbon moieties [36,37].In intervention studies, higher intake of choline did also not alter IGF2 methylation [34] and the use of SQLNS did not affect methylation of IGF1, another major growth factor [33].This is in contrast with other studies showing that maternal intake of methyl-group donors including folate/folic acid associated negatively with IGF2 methylation in buccal epithelial cells in 6 months old children and positively with IGF2 methylation in infant blood and cord blood suggesting a tissue-specific response [15,54,55].
LEP encodes the hormone Leptin which plays a central role in energy homoeostasis including appetite regulation and mutations in LEP can contribute to the development of obesity and diabetes type 2 [56].Different results for LEP methylation have been reported depending on studied onecarbon moiety: positive associations with serum folate, negative associations with serum vitamin B 12 [44], and no associations with choline have been reported [37] (Both ErasmusAGE score 6).
Cortisol regulating genes could play a role in multiple diseases including cardiovascular complications.Jiang et al. investigated choline intake (480mg/ daily versus 930 mg/daily) and found increased methylation in the higher dose group of the CRH promotor and the 5'untranslated exon 1F of NR3C1 [4].In contrast, NR3C1 methylation associated negatively with choline levels in a study by Nakanishi et al. (ErasmusAge score 6) [37].A possible explanation is that both studies targeted a different genomic region of NR3C1 or there may be lack of comparability between blood levels of choline and use of choline supplements.
Nakanishi et al. investigated several other genespecific sites related to growth and metabolism and choline in maternal blood and cord blood.Negative associations with choline levels were also found for AdipoQ, PPARG1A, HSD11β2, PPARA, NDUFB6, and RXA, while no associations were found with methylation of MEG3 and MEST, all involved in foetal growth.Differences have been found for different time points during pregnancy [7].In an in vitro study, MEST methylation was also not altered in response to folic acid [51].
MMP-9 codes for a matrix metalloproteinase induced during labour and MMP-9 is also increased in multiple cardiovascular diseases including hypertension and myocardial infarction [57].Serum vitamin B 12 and folate associated positively with MMP-9 promoter methylation and with lower MMP-9 RNA and protein levels [41].Contrary, folic acid treatment did not change MMP-9 methylation in an in vitro model [43].

Genes related to placental development and function
The one-carbon metabolism has also been linked to placental angiogenesis which is crucial for a well-functioning placenta.The placenta itself produces several angiogenic factors involved in endothelial growth and function, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), both of which are decreased in preeclampsia (PE) [58,59].For example, increased maternal choline intake can suppress anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT-1) expression in human placentas and promotes angiogenesis in mice placentas [10].Vitamin B 12 supplements did not alter VEGF methylation in a high-quality intervention study [30], while another study showed PlGF methylation associated with SAM and SAM:SAH in the placenta [39].Since a higher SAM:SAH ratio indicates higher availability of methyl group donors (Figure 1), a deranged one-carbon metabolism may be involved in the pathogenesis of placentalrelated complications partly through inducing epigenetic changes resulting in impaired placental functioning.

Genes directly related to one-carbon metabolism
Plasma vitamin B 12 was negatively associated with MTR promoter methylation.This can have an impact on the one-carbon metabolism, since MTR codes for methionine synthase which is crucial in the conversion of homocysteine to methionine [40] (Figure 1).

Genes selected based on prior genome-wide analysis
Lastly, Liu et al. selected gene-specific sites of interest based on DMRs identified through genome-wide analysis.Methylation of FAM198A associated negatively with preconceptional start of folic acid only in male offspring indicating a sex-specific impact on DNA methylation.No associations were found between timing of folic acid supplements and methylation of 2 ANKRD20B CpGs [35].
In summary, most studies reported both significant and non-significant associations depending on the studied genes and moieties, indicating a sitespecific relationship between the one-carbon metabolism and DNA methylation at various genes in the placenta (Table 2).

Previous Reviews
Our review is largely in line with previously published human and animal reviews.James et al. focused predominantly on DNA methylation in cord blood and showed several associations between maternal one-carbon metabolism and methylation of specific genes.Overlapping genes with the current review include IGF2/H19, LEP, NR3C1, RXRA, MEG3, MEST, IL10, and GNAS-AS1 and several other associations were reported.For most genes, differences were found depending on studied tissue, moiety, specific loci, and timing of exposure [12].In rodents, maternal folic acid supplements [24] and choline intake or methyl group supplemented diets [14] have been shown to impact DNA methylation at several genespecific sites, leading to both hypo-and hypermethylation depending on studied genomic region.Additionally, these studies found mixed outcomes for global methylation in multiple offspring tissues including brain, mucosa, and liver.For homocysteine, associations with hypomethylation were more prominent in animal models as compared to human studies, where mixed results have been reported [25].Likewise, multiple studies using livestock showed that dietary restrictions or excesses, or a methyl supplemented diet can affect both gene-specific and global DNA methylation in the offspring, depending on studied tissue, offspring sex, and timing of exposure [26,60].A systematic review focusing on physical activity and dietary intake of carbohydrates, fats, and proteins during pregnancy showed similar mixed associations with DNA methylation in placental tissues and cord blood [61].This could indicate a role for a variety of lifestyle behaviours affecting the epigenome, however, the one-carbon metabolism is influenced by lifestyle behaviours which could also be (part of) the underlying biological mechanism for observed associations.

Strengths and Limitations
In the present article, we systematically review studies investigating relationships between different one-carbon moieties and DNA methylation focusing on the human placenta.We included a wide range of exposures and outcomes and were not limited to a specific timing in relation to delivery, to be able to give a comprehensive overview of current literature.Consequently, heterogeneity between studies limits comparability of results and a meta-analysis is therefore not possible.Besides, most included studies face several challenges which should be taken into account.First, dietary patterns and vitamin use are often associated with other (lifestyle) behaviours potentially affecting DNA methylation, including smoking, BMI, and socio-economic status.Most studies are observational and do not adequately adjust for potential confounders.Second, one-carbon moieties are usually not consumed independently of each other and of other nutrients in the diet, creating difficulties when studying the effect of a single moiety.Additionally, most studies were performed in healthy cohorts without major nutritional deficiencies.The effect of differences in onecarbon moieties could be limited within a normal range.Sample size of most included studies is relatively small and self-reported exposures like food questionnaires and vitamin intake could be less reliable.Differences in sampling methods and storage conditions of placental tissues could also lead to differences between studies.Lastly, the timing between exposure and measuring outcome may affect results.Placental DNA methylation is only studied postpartum in all included studies, and might not accurately reflect methylation profiles at time of earlier exposures because of accumulating exposures during gestation also impacting DNA methylation profiles [62].

Clinical Implications and Future Research
Current studies investigating one-carbon metabolism and placental DNA methylation display low overlap in terms of studied exposures and genomic regions, and the number of high-quality intervention studies is limited.Therefore, high-quality intervention studies in combination with a genome-wide readout for DNA methylation and preferably randomized and blinded, are warranted to further investigate the impact of an optimized maternal one-carbon metabolism on DNA methylation profiles and health in the offspring.Ideally, studies will include follow-up after birth to assess the impacts on lifelong health.Furthermore, DNA methylation is highly cell type specific.Investigating placental DNA methylation per cell type could therefore decrease heterogeneity both within and between studies.Moreover, placental tissue might not be an appropriate model for some research questions and other tissues or cell types could also be incorporated [63]; however, tissues from internal organs are largely inaccessible and the placenta could provide insight in both placental function and foetal epigenetic programming.Generally, placental DNA methylation is only studied at one time point, i.e., after delivery.In the future, the presence of cfDNA originated from placental cell types derived from maternal blood might be an unique in vivo opportunity to non-invasively study placental DNA methylation profiles already during pregnancy [23].Moreover, cfDNA could potentially provide targets to improve future prenatal diagnosis and obstetrical care by identifying modifiable epigenetic derangements during pregnancy, contributing to improved health outcomes across the life course.

Conclusions
Most included studies indicate relationships between several measures of one-carbon moieties in the prenatal period and site-specific DNA methylation in the placenta rather than changes in global DNA methylation.Several gene-specific associations with DNA methylation were found, especially in genes involved in growth and metabolism.A deranged one-carbon metabolism leading to detrimental changes in the foetal epigenome in genes involved in for example the cardiovascular system may support the DOHaD paradigm.Changes in DNA methylation could not only lead to altered placental functions but could also serve as a proxy for foetal epigenetic programming, with both consequences for foetal development as well as future health outcomes.
On the other hand, conflicting outcomes were reported and there is a lack of uniformity between studies in geographically study populations, investigated loci, exposure measures, and study designs.Moreover, DNA methylation is only studied in postpartum placentas, though this may be overcome by investigating methylation of cfDNA in future studies.High-quality intervention studies tackling common limitations of included studies are needed to elucidate the relationship between one-carbon metabolism and specific differences in epigenetic programming in the offspring, ideally with longitudinal follow-up after birth.This could enable us to assess the corresponding impact on health outcomes across the life course and identify preventative or therapeutic measures contributing to improved health outcomes for future generations.

Figure 1 .
Figure 1.Simplified overview of the one-carbon metabolism.Dietary methionine is converted to homocysteine via SAM and SAH.SAM is a major methyl donor for biological processes, including DNA methylation.The remethylation of homocysteine to methionine depends on other moieties including vitamin B12 as cofactor and 5 methyl tetrahydrofolate as substrate.MTHFR = methylenetetrahydrofolate reductase, BHMT = betaine-homocysteine S-methyltransferase, MS = methionine synthase, SAM = s-adenosylmethionine, SAH = s-adenosylhomocysteine

Figure 2 .
Figure 2. The one-carbon metabolism as an underlying pathway affecting placental DNA methylation and lifelong health outcomes of the offspring.Maternal exposures such as nutrition and the use of vitamin supplements, are involved in the one-carbon metabolism, which provides methyl groups for DNA methylation processes (left).DNA methyltransferases (DNMTs) transfer methyl groups to the cytosine-phosphate-guanine sides (CpGs).Derangement of the one-carbon metabolism are proposed mechanisms affecting DNA methylation processes (bottom).The placenta is the intermediate between the maternal environment and embryonic and foetal growth and development.Epigenetic derangements can affect (1) directly the functioning of the placenta and subsequently foetal growth and development, and (2) the foetal epigenome with potential consequences for foetal growth and development, and 1 and 2 both have consequences for lifelong health outcomes in the offspring.

Table 1 .
Study characteristics and main findings of included studies ordered according to the quality score.
in vitamin B 6 , folate, and betaine but low in vitamin B 12 showed a significant negative association with 2 DMRs related to DLL1 (p = 5.3.10 −7 , p = 7.0.10−9 ) and FAR1 (p = 3.7.10−9 , p = 7.8.10 −3 ).Lastly, 'bread and starchy food' diet which is rich in betaine but low in vitamin B 2 , B 6 , [32].After adjustment for vitamin use, the 'varied and balanced' diet which is rich in B vitamins, choline, and methionine but low in betaine showed a positive association with a DMR related to NPDC1 (p=0.02).A 'vegetarian tendency' diet which is rich

Table 2 .
Direct associations between one-carbon moieties and global methylation, genome-wide methylation or gene-specific methylation in included clinical studies with an ErasmusAGE quality score of at least 5 out of 10 points.